INTRODUCTION

Top Abstract Introduction Materials and Methods References

In eukaryotic cells, organelles of the secretory pathway are interconnected by dynamic vesicular traffic. From the endoplasmic reticulum (ER), a variety of proteins are transported to the Golgi apparatus by carrier vesicles and then to their final destinations. On the other hand, in order to maintain the ER-specific functions, many resident ER proteins have to be correctly retained in the ER. Two mechanisms of ER localization have been proposed. One is static retention, which prevents ER proteins from being exported from the ER, and the other is dynamic retrieval, which returns ER proteins from the Golgi apparatus to the ER (12, 14, 15).

The Saccharomyces cerevisiae RER2 gene was originally identified as being involved in this ER protein localization (12). We isolated mutants of two complementation groups that mislocalize an ER membrane protein, Sec12p, and designated them rer1 and rer2 (for retention or retrieval of ER proteins). The rer2 mutants were temperature sensitive for growth and showed pleiotropic phenotypes: in addition to ER protein mislocalization, they showed slow growth, defects in N- and O-glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and Golgi apparatus (16). The rer2 disruptant could grow very slowly, but the double disruption of RER2 and SRT1, a homologue of RER2, was a lethal event (16).

In the course of our study of the RER2 gene, we found that RER2 homologues exist not only in eukaryotes but also in prokaryotes. In Escherichia coli, the RER2 homologue had been registered in the genome sequence as a hypothetical open reading frame, o253. We designated it rth (for RER2 homologue) and decided to investigate its biological function to obtain a clue to the roles of the RER2 gene family. In this paper, we will present evidence that the E. coli rth gene encodes undecaprenyl diphosphate synthase (UDS), which is responsible for the synthesis of undecaprenyl phosphate, the carrier lipid required for the biosynthesis of peptidoglycan in the cell wall. In parallel, we showed that the yeast Rer2 protein is required for the synthesis of dolichol, the polyprenyl compound involved in protein glycosylation (16). The conserved essential function of the Rer2 family as cis-prenyltransferase will be discussed.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods References

Materials. [1-¹⁴C]isopentenyl diphosphate (IPP) (specific activity, 2.04 TBq/mol) was purchased from Amersham Co. Nonlabeled IPP and all-E-farnesyl diphosphate (FPP) were synthesized by phosphorylation of the corresponding prenols (9). Solanesol (all-E-nonaprenol) and dodecaprenyl phosphate were purchased from Sigma Chemical Co. Ficaprenols were provided by Nisshin Flour Milling Co. Thin-layer chromatography (TLC) plates (silica gel 60 F₂₅₄ and LKC-18) were purchased from Merck Co. and Whatman Co., respectively.

Bacterial strains, plasmids, and culture media. The E. coli strains used were MG1655 (rph ilvG rfb) (2), DH1 (recA1 endA1 gyrA96 thi-1 hsdR17 supE44) (13), and N1126 (thi leu pro malA his thy cysC lacZ ara mtl xyl str spc polA12) (19). The plasmids used were pUC19 (20), pUC4K (20), pBR322 (13), pJK274 (11), pJK282 (11), and pJK286 (10).

Plasmid construction. For cloning of the rth cdsA region, the HindIII DNA fragments containing the rth⁺ and cdsA⁺ genes were prepared by PCR with the oligonucleotides 127-1 (5'-CCAAGCTTCTGCGGACGTCTGTTTATGG-3') and 127-2 (5'-CCAAGCTTAAACGCTCAACGCGAACACC-3') as primers and the rth⁺ cdsA⁺ BamHI fragments were prepared with the oligonucleotides 127-3 (5'-CCGGATCCCTGCGGACGTCTGTTTATGG-3') and 127-4 (5'-CCGGATCCAAACGCTCAACGCGAACACC-3') as primers. MG1655 cells were used directly as templates, and Takara Ex Taq polymerase (Takara Shuzo Co., Kyoto, Japan) was used as the enzyme. The rth⁺ cdsA⁺ HindIII fragment was inserted into the HindIII site of pUC19, and the SmaI Cm^r cassette of pJK211 was inserted into the resultant plasmid at the HpaI site in the rth gene to obtain pUC19-rth::Cm^r cdsA⁺. pJK211 was constructed by inserting the BamHI Cm^r cassette of pJL3-4779 (11), after treatment with Klenow fragments, into the HincII site of pUC8 and by converting the HindIII site of pUC8 into a SmaI site by using Klenow fragments and a SmaI linker. The rth⁺ cdsA⁺ mini-F plasmids were constructed by inserting the rth⁺ cdsA⁺ HindIII PCR fragment into the HindIII site of pJK286 (mini-F Ap^r) and inserting the rth⁺ cdsA⁺ BamHI PCR fragment into the BamHI site of pJK282 (mini-F Km^r). The rth⁺ cdsA⁺ BamHI fragment was also inserted into the BamHI site of pJK2039 to obtain pJK2039-rth⁺ cdsA⁺. A multicopy vector, pJK2039, was constructed by ligating the PvuII Km^r fragment of pJK274 (11) with the NruI-ScaI fragment of pBR322. The SmaI Cm^r cassette of pJK211 was inserted into the HpaI site in the rth gene of pJK2039-rth⁺ cdsA⁺ to obtain pJK2039-rth::Cm^r cdsA⁺. The PstI fragment containing both the Km^r marker and a part of the cdsA gene of pJK2039-rth⁺ cdsA⁺ was replaced with the Km^r PstI fragment of pUC4K to construct pJK2039-rth⁺ cdsA.

Disruption of the rth gene. For disruption of the rth gene, the E. coli polA12 strain, N1126, was first transformed with pJK282 (mini-F Km^r)-rth⁺ cdsA⁺. The cells were further transformed with pUC19-rth::Cm^r cdsA⁺ and then incubated at 42°C. Among the Cm^r transformants, Ap^s colonies that were devoid of the pUC plasmid were selected. By eliminating the strains with the Cm^r marker inserted into mini-F plasmids, we obtained the desired rth disruptant strain, N1126 rth::Cm^r/pJK282-rth⁺ cdsA⁺. To construct MG1655rr (rth::Cm^r recA::Tn10)/pJK282-rth⁺ cdsA⁺, MG1655 was transformed with pJK282-rth⁺ cdsA⁺ and the rth gene of the resultant strain was disrupted by P1 transduction with N1126 rth::Cm^r/pJK282-rth⁺ cdsA⁺ as a donor. The recA gene was inactivated by P1 transduction with GL787L as a donor (4).

Isolation of temperature-sensitive mutants. The rth(Ts) mutants were isolated by plasmid shuffling with a mini-F plasmid as a vector (10). The rth cdsA region was mutagenized by error-prone PCR in the presence of 0.2 or 0.4 mM MnCl₂ (21) with the primers 127-1 and 127-2 and the plasmid pJK282-rth⁺ cdsA⁺ as the template. The amplified HindIII fragments were cloned into a mini-F vector, pJK286 (Ap^r), and the resultant plasmids were introduced by electroporation into the rth disruptant, MG1655rr/pJK282 (Km^r)-rth⁺ cdsA⁺. Among the Ap^r transformants, temperature-sensitive mutants were selected on antibiotic medium 3 plates containing ampicillin at 42°C and then confirmed for kanamycin sensitivity at 30°C to make sure that the original Km^r mini-F plasmid was absent.

Preparation of enzyme fractions. Cells from a 100-ml culture were disrupted in 2 ml of 100 mM potassium phosphate (pH 7.4) and 10 mM 2-mercaptoethanol with a Tomy UD-200 ultrasonic disintegrator eight times for 15 s at 30-s intervals. After centrifugation, a supernatant fraction was obtained and used as a cell homogenate.

Prenyltransferase reaction and product analysis. The reaction mixture contained, in the final volume of 0.2 ml, 2.0 nmol of [1-¹⁴C]IPP (1.1 × 10⁵ dpm; specific activity, 0.92 TBq/mol), 1.0 nmol of FPP, 0.2 µmol of MgCl₂, 0.2 mg of Triton X-100, 10 µmol of potassium phosphate (pH 7.5), and the enzyme fraction. After incubation at 30 or 40°C for 30 min, the reaction was stopped by heating it at 95°C for 3 min.

Extraction of isoprenoids. Isoprenoids were extracted from cells as described previously (8), with a slight modification. About 10 mg (dry weight) of cells was harvested by centrifugation and washed once with 0.85% NaCl. After the NaCl solution was removed as completely as possible, the cells were suspended in 2 ml of methanol. Solutions of ubiquinone-10, phylloquinone, and dodecaprenyl phosphate were added to the suspension as internal standards for isoprenoid quantitation. Isoprenoid quinones were extracted from this mixture with hexane. One milliliter of 60% KOH was added to the residual methanol-water layer, and the mixture was heated in a boiling-water bath for 60 min. The hydrolysis products, polyprenyl monophosphates, were extracted with diethyl ether. The diethyl ether extract was washed with 5% acetic acid and dried under N₂.

Analysis of isoprenoids. The hexane extract containing isoprenoid quinones was loaded on a 0.4-g column of neutral alumina (grade III). Menaquinone and demethylmenaquinone were eluted with 2.5% diethyl ether in hexane, and ubiquinone was eluted with 7.5% diethyl ether in hexane. These compounds were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase octyldecyl silane (ODS) column (Hitachi 3056; 4.0 by 150 mm; eluent, 2-propanol-methanol, 1:1 [vol/vol]; flow rate, 1 ml/min). Menaquinone and demethylmenaquinone were monitored at 248 nm with a spectrophotometric detector, and their amounts were calculated from the ratio of the peak area to the internal standard, phylloquinone. Ubiquinones were monitored at 275 nm, and the ratio of the peak area to the internal standard, ubiquinone-10, was used for the quantitation.

	RESULTS AND DISCUSSION

rth is essential for cell growth. The E. coli gene rth (open reading frame o253 [3]), which is located at 4.2 min, encodes a protein of 253 amino acid residues which exhibits extensive homology to the yeast Rer2 protein (Fig. 1A). The yeast rer2 mutant showed interesting pleiotropic phenotypes, including ER protein mislocalization, glycosylation deficiency, and accumulation of abnormal membranes. Although these phenotypes are unique to eukaryotes, the family of genes is evolutionarily conserved from prokaryotes to higher eukaryotes. Therefore, we were puzzled by but interested in the functions of this gene family. To obtain a clue to their biological roles, we decided to examine the function of the E. coli counterpart, rth.

View larger version (34K): [in this window] [in a new window]   FIG. 1.   E. coli rth gene. (A) Homology between the S. cerevisiae Rer2 protein and the E. coli Rth protein. The polypeptides of Rer2 and Rth are shown as boxes. The shaded and striped boxes represent homologous domains, and the percent values indicate identity and similarity (in parentheses) between the two domains. The numbers along the polypeptides indicate the amino acid (aa) residue numbers. (B) Chromosomal inserts in plasmids used for complementation test. The arrows indicate open reading frames in the rth region. The arrowheads represent the PCR primers used for cloning. The chromosomal regions cloned into the vector pJK2039 are shown as three open boxes: top, the PCR fragment amplified with primers 3 and 4; middle, the same PCR fragment with a Cmr cassette inserted into the HpaI site within the rth gene; bottom, the PCR fragment lacking the 3' region of the cdsA gene. Plus and minus signs denote complementation, positive and negative, respectively, for both rth-2 and rth-6 mutations.

rth mutants show a defect in cell wall rigidity. We examined the growth of the rth mutant strains at permissive and nonpermissive temperatures. Typical growth curves of rth⁺ and rth-6 cells at 30 and 42°C are shown in Fig. 2. As shown in Fig. 2A, the growth of the rth-6 mutant was normal at 30°C but significantly retarded at 42°C in LB broth containing 0.5% NaCl. The temperature-sensitive growth was much more evident when the rth-6 mutant was cultured in LB broth containing no NaCl. The mutant cells grew almost normally at 30°C but did not grow at all at 42°C (Fig. 2B). The rth-2 mutant showed growth profiles very similar to those of the rth-6 mutant at both 30 and 42°C. The rth-4 mutant grew more slowly than the other mutants even at 30°C, perhaps due to the additional cdsA mutation (data not shown). These results indicate that the rth(Ts) mutants are sensitive to low osmolarity at high temperature.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Growth of rth+ and rth-6 cells. Strains MG1655rr/pJK286-rth+ cdsA+ and MG1655rr/pJK286-rth-6 cdsA+ were grown in LB broth containing 0.5% NaCl at 30°C for 12 h. Then, 0.05 ml of these cultures was inoculated into LB broth containing 0.5% NaCl (A) or without NaCl (B). The cells were grown at 30 or 42°C with shaking. Optical densities at 660 nm (OD660) were measured at 1-h intervals. Open squares, rth+ at 30°C; solid squares, rth+ at 42°C; open circles, rth-6 at 30°C; solid circles, rth-6 at 42°C.

View larger version (104K): [in this window] [in a new window]   FIG. 3.   Cell shapes of the rth-6 mutant. The upper panels show the rth+ strain, MG1655rr/pJK286-rth+ cdsA+; the lower panels show the rth-6 mutant, MG1655rr/pJK286-rth-6 cdsA+. The cells were incubated at the indicated temperatures for 2 h and observed without fixation under a microscope equipped with phase-contrast optics. The magnifications are the same for all of the panels (a 100× objective was used). The arrowheads in the panel with the rth mutant incubated at 42°C indicate ghosts.

rth mutants are defective in UDS activity. In the course of this study, we realized that the E. coli Rth is also homologous with a Micrococcus luteus protein which was very recently reported to have the activity of UDS in vitro (17). This enzyme is a cis-prenyltransferase that adds isoprene units onto FPP to produce undecaprenyl (C₅₅) diphosphate, the precursor of the carrier lipid for peptidoglycan synthesis (Fig. 4).

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Biosynthesis of isoprenoids.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Prenyltransferase activity. (A) Prenol products synthesized in an in vitro assay. Cell homogenates were prepared from the strains MG1655rr/pJK286 (mini-F Apr)-rth+ cdsA+ (lanes 1 and 2), MG1655rr/pJK286-rth-2 cdsA+ (lanes 3 and 4), MG1655rr/pJK286-rth-4 cdsA(Ts) (lanes 5 and 6), and MG1655rr/pJK286-rth-6 cdsA+ (lanes 7 and 8). The homogenates (0.1 mg of protein) were incubated with 10 µM [1-14C]IPP and 5 µM FPP in a final volume of 0.2 ml at 30°C (lanes 1, 3, 5, and 7) or at 40°C (lanes 2, 4, 6, and 8) for 30 min. The resulting prenols were extracted and analyzed by TLC on a silica gel 60F254 plate with a benzene-ethyl acetate (4:1 [vol/vol]) solvent system and by autoradiography. For more details, see Materials and Methods. The arrowheads indicate the positions of authentic prenols: Fi, ficaprenols; So, solanesol (all-E-nonaprenol); Ori, origin; SF, solvent front. (B) Fractionation of cell homogenates. The homogenates were prepared from the same strains shown in panel A, rth+ (open circles), rth-2 (solid triangles), rth-4 (solid squares), and rth-6 (solid circles), and fractionated by DEAE-Toyopearl column chromatography. The prenyltransferase assay was carried out at 30°C with 50 µl of each fraction as the enzyme source in a final volume of 0.1 ml. The dotted line indicates the NaCl concentration of the elution buffer. (C) Prenol products of fraction 3. The cell homogenates prepared from the strains shown in panels A and B, rth+ (lanes 1 and 2), rth-2 (lanes 3 and 4), rth-4 (lanes 5 and 6), and rth-6 (lanes 7 and 8), were fractionated as described for panel B, and fraction 3 of each sample was incubated with [1-14C]IPP and FPP in the presence of 0.04 mg of Triton X-100 in the 0.2-ml mixture. The reaction was carried out at either 30 (lanes 1, 3, 5, and 7) or 40°C (lanes 2, 4, 6, and 8) for 30 min (lanes 1 and 2) and 3 h (lanes 3 to 8). The resulting prenols were analyzed by TLC on a reversed-phase LKC-18 plate with an acetone-water (19:1 [vol/vol]) solvent system and autoradiography. The arrowheads indicate the positions of authentic prenols: So, solanesol (all-E-nonaprenol); C50, decaprenol (ficaprenol-50); C55, undecaprenol (ficaprenol-55); C60, dodecaprenol (ficaprenol-60); Ori, origin; SF, solvent front.

Undecaprenyl phosphate is decreased in rth mutant cells. In order to discover the effects of the rth mutations on isoprenoid synthesis in vivo, we further measured the cellular levels of isoprenoids in the mutants. Isoprenoids were extracted from the cells cultured at 30°C and were analyzed by HPLC. As shown in Table 2, the levels of polyprenyl phosphates, particularly undecaprenyl phosphate, were appreciably lower in the mutants than in the wild-type cells, even though the cells were cultured at the permissive temperature. On the other hand, the levels of isoprenoid quinones were not significantly changed or were even higher in the mutants. These results indicate that Rth is indeed responsible for the formation of undecaprenyl phosphate in vivo. It should be noted here that the remaining activities to synthesize polyprenyl phosphates at 30°C are considerable in the mutants in contrast to the in vitro results of the UDS activities (Fig. 5). The major products of the enzyme were C₅₀, C₅₅, and C₆₀ phosphates in the mutant cells, unlike the in vitro results. These observations suggest that the UDS enzymes from the mutants were less stable in vitro, perhaps due to the lack of other cellular components, and show more profound defects in vitro than in vivo. At 42°C, the cellular level of undecaprenyl phosphate was further decreased in the mutants. However, when the cells were incubated at 42°C, the HPLC profiles showed an unidentified peak near dodecaprenyl phosphate, which made the correct quantitation of polyprenyl phosphates difficult (data not shown).

Conclusions. The results in this work have clearly shown that Rth is responsible for prenyl chain elongation in carrier lipid synthesis in vivo. From this, together with the fact that Rth is structurally homologous to the Micrococcus UDS, we conclude that Rth functions as the cis-prenyltransferase, UDS, for peptidoglycan synthesis. The development of this work greatly stimulated our study of the yeast Rer2 protein, and in fact, we have now demonstrated that the rer2 mutant is deficient in the yeast cis-prenyltransferase activity required for dolichol synthesis and concluded that the RER2 gene encodes this key enzyme (16). Dolichol is essential for N- and O-linked protein glycosylation in yeast, but interestingly, not all the phenotypes of the rer2 mutant can be explained by the glycosylation deficiency, suggesting a novel physiological role of dolichol.